Abstract

Being an immobile Gram-positive soil bacterium, Corynebacterium glutamicum has to cope with dramatic changes of the environmental conditions like an altered osmolarity or temperature. It was recently shown, that the MtrB-MtrA two-component system of this bacterium mediates the expression regulation of osmo- and chill stress-related genes in response to an increased medium osmolarity or decreased environmental temperature. The response regulator of this system, MtrA, was shown to act as an activator of the genes proP, betP, and lcoP as well as a repressor of the mscL gene. These results led to the question, which physico-chemical signals are recognized by MtrB, in order to mediate the expression regulation of genes in response to changes of the environmental osmolarity and temperature. To avoid the high complexity of the cellular system, an in vitro system was established for the detailed analyses of the sensory properties of MtrB. Using E. coli liposomes enriched with MtrB, the basic characteristics of the bacterial two-component system could be detected. These included the autokinase activity of membrane-bound MtrB, the phosphoryl group transfer to soluble MtrA, and the MtrB-catalyzed dephosphorylation of MtrA-P. By use of the artificial membrane system the influence of a systematic variation of signals related to osmo- and chill stress conditions could be performed. The autokinase activity of MtrB turned out to be significantly stimulated by the presence of numerous osmolytes including sugars, amino acids, compatible solutes, and high molecular weight PEGs. In addition, the histidine kinase was shown to be strongly activated by exposure to low temperature. By fusion of proteoliposomes made from E. coli lipid with synthetic POPG, the sensory properties of MtrB furthermore turned out to significantly depend on the composition of the membrane surrounding. In order to detect the sensing domain of MtrB, various truncated derivatives of the protein were constructed. In contrast to most bacterial histidine kinases, which are thought to exhibit their sensory function within the extracytoplasmic loop, MtrB was shown to sense independently of this external domain. Taken together, the data of this work led to the model, that MtrB senses environmental hyperosmotic stress either as an increased osmolarity of the cytoplasm, or as altered membrane properties. Environmental chill conditions are probably sensed as an altered physical state of the membrane.